1. Field of Invention
The present invention relates to a composition comprising an immortalized cell line which is deficient in the expression of one or more gene(s) of the intrinsic apoptotic pathway. In particular, said cell line is on deposit and publicly available through the Center for Technology Transfer of the University of Pennsylvania, said cell line having docket number O-2707. The present invention further relates to the product of a process for producing an immortalized mouse fibroblast cell line which is deficient in the expression of one or more of Bax, Bak, or Bax and Bak gene(s). Finally, the present invention relates to a method and a kit for identifying a compound or composition which induces or blocks the extrinsic apoptotic pathway.
2. Background
Programmed cell death, known as apoptosis, is a process where a cell kills itself in response to extrinsic signals, or intrinsic or developmental cues. Organisms use apoptosis to eliminate damaged or unwanted cells. Apoptosis plays a critical role in development and tissue homeostasis. In humans and other mammals, excessive apoptosis may lead to tissue degenerative diseases such as Alzheimer's disease, while insufficient apoptosis leads to uncontrolled cell growth that may result in cancer. Two major apoptotic pathways have been identified: an extrinsic cell death pathway in which apoptosis is initiated through ligand binding to cell surface receptors expressed on the cell that will subsequently die, and an intrinsic cell death pathway in which apoptosis is initiated within the cell.
Tumor necrosis factor receptor family members are among the best characterized of receptors involved in the extrinsic death pathway. Upon the engagement of ligand, these receptors initiate the formation of a death-inducing signaling complex, which includes an essential adaptor molecule FADD. FADD in turn recruits Caspase 8, which in turn is activated through an autoproteolytic process. Once activated, Caspase 8 initiates both the activation of additional caspases such as Caspase 9 and Caspase 3, and the degradation of intracellular substrates, which eventually results in cell death. The TNF receptor family includes TNFR1; Fas; DR3 proteins such as Apo3, WSL-1, TRAMP, and LARD; DR4; DR5 proteins such as TRAIL-R2, TRICK2, and KILLER; and DR6.
Members of the Bcl-2 family of proteins are characterized by their ability to modulate cell death. Bcl-2 and some of its homologues, such as Bcl-xl, inhibit apoptosis, whereas other family members, such as Bax and Bak, induce or accelerate apoptosis under certain conditions. Bak and Bax, as well as Bcl-xs, Bid, and Bik, constitute the pro-apoptotic group of Bcl-2 proteins.
The Bax protein shares highly conserved domains with Bcl-2, some of which are required for the formation of Bax/Bcl-2 heterodimers, which are thought to be important for the survival or death response to apoptotic signals.
Bax expression is elevated in certain tissues after apoptotic stimuli and can be directly regulated by p53. Bax can form ion-conducting channels in lipid bilayers, such as mitochondrial membranes, which may be the biochemical mechanism through which it exerts some of its effects.
Bax was first identified as a protein that co-immunoprecipitated with Bcl-2. Determination of the amino acid sequence of the Bax protein showed it to be highly homologous to Bcl-2. The bax gene encodes a 21 kDa protein which is 96% homologous between mouse and human.
Bak also promotes cell death and counteracts the protection from apoptosis provided by Bcl-2. Like Bax, the Bak gene product primarily enhances apoptotic cell death following an appropriate stimulus. Bak is a potent inducer of apoptosis in various cell types. The predicted mouse Bak protein is 77% identical and 89% similar to its human counterpart.
U.S. Pat. No. 6,245,885, issued Jun. 12, 2001 to Shore, et al., discloses purified polypeptide fragments of the ART domain and the transmembrane domain of the BAX protein which, when administered to a cell, increase or decrease apoptosis of the cell. Also disclosed are methods for identifying compounds which, when administered to a cell, increase or decrease apoptosis of the cell. Also disclosed are methods for diagnosing a patient having, or predisposed to develop, a disease involving altered apoptosis by identifying a mutation in a BAX-encoding gene which results in an amino acid mutation in the BAX ART domain, a BAX transmembrane domain, or that alters the interaction of the BAX ART domain with the BAX transmembrane domain.
To study how the apoptotic machinery is regulated, mice deficient in both Bax and Bak have been produced. In line with the importance of Bax and Bak in apoptosis, such mice show developmental defects in multiple tissues. However, because of the difficulty in breeding Bax/Bak double deficient mice, and the inability of primary cells to proliferate infinitely, the use of primary lymphocytes or fibroblasts to study molecular events of apoptosis is limited.
To satisfy this need in the art, Applicants have developed the inventive immortal mouse embryo fibroblast cell lines, which are Bax/Bak double deficient, Bax deficient, and Bak deficient. Such deficient cell lines are resistant to wide variety of intrinsic death stimuli. The establishment of the inventive immortal cell line provides an unlimited source of Bax and/or Bak deficient cells. The inventive cells are more homogeneous and stable than primary cells. They do not generally change their growth properties during further culturing and propagation, and they have a defined genetic background. They are easy to transfect: unlike primary fibroblasts, where expression vectors can only be transduced through retroviral infection, the inventive immortal cells can be transfected using various standard methods.
The availability of the inventive immortal cell lines will make possible the study of the molecular events of apoptosis which could not previously be studied directly, such as the mechanisms by which these proteins regulate the cell fate of death or survival and how the activities of these proteins are regulated by different survival or death-inducing signals. Further, the inventive cell lines and methods for using the same will make it possible to investigate whether a drug is toxic to cells or protects cells from dying in a mechanism-based manner, as well as to identify and evaluate non-apoptotic side effects.